Chapter 3: Gene expression (lecture 1) Flashcards
What will be discussed in the lecture? (you obv don’t have to learn this, just so you know what to expect)
- Transcription
- Chromatin structure
- DNAmethylation
- microRNAs (miRNAs)
- Telomeres and telomerase
- Therapeutic options
What are the three steps as the promotor site that need to occur for initiation of transcription?
- Transcription factor binds to response element
- RNA polymerase II (holo-enzyme) binds to TATA-box
- Transcription starts
For indication: how many transcription factors are there?
About 3000 in humans (so 1 out of 6 genes)
Transcription factors (TF) recognize a specific DNA sequence. How is this sequence called?
Response element
What are co-activators/suppressors and what do they do?
Accessory/supportive molecules that interact with the DNA-binding proteins to promote/suppress RNA transcription
Other regulatory elements are enhancers and silencers. Where are they located and what do they interact with?
They are located outside the promoter region (can be up- or downstream). They can be bound by regulatory proteins and interacting co-factiors
Transcription factors (TF) activity can be regulated by five different ways. What are these five?
- Dimerization
- Ligand binding
- Expression restricted to particular cell types
- Covalent modifications (phosphorylation)
- Cellular localization
The transcription factors (TF) consist of various regions, some regions they all have, some regions only some have. Can you name them?
All TFs:
1) DNA binding domain
2) Transcriptional activation domain (binding of other components of the transcription machinery (pol II))
Some TFs (domains to control activity of TF):
3) Dimerization domain
4) Ligand binding domain
What are examples of DNA-binding domains? (you don’t have to know each of them)
- Zinc finger
- Helix-loop-helix
- Helix-turn-helix
- Leucine zipper
What protein is involved in the ligand binding domains for the regulation of TF activity?
The steroid hormone receptor superfamily (it has 48 members)
Can you name an example of the steroid hormone receptor superfamily that influences TF activity? (You don’t actually have to know this, but it’s a good example to see what happens in the cell)
Retinoic acid (vit A) receptor (RAR), represses transcription in the absence of RA (left figure). The book also gives an example for glucocorticoid that does not directly into the cell, but first binds to form a complex.
Another example of TF activity that was mentioned is dimerization domains. How does this work?
Some tissue factors can interact with other tissue factors, forming a dimerization. This can either be a homo- or heterodimer. Remember that the figure is a very simplified explanation
What are two common tissue factor families that form dimerization domains?
AP-1: Members of the Jun and Fox TF family. There are 18 possible combinations (jun-for heterodimers and jun homodimers). Jun = pro-proliferative, Jun B = anti-proliferative
Another interesting feature is that domains can function independently. Explain this by the example of the steroid hormone receptor superfamily. (in advance, sorry for the amount of text, but I wanted to keep the info to one slide to show the full picture, just make sure you understand this and look closely to the figure)
The steroid hormone receptor family contains a zinc finger type of DNA-binding domain, a ligand-binding domain for a specific steroid hormone, and a dimerization domain. Each domain functions independently (=specific for a particular steroid hormone receptor). When we looking at the figure, if the ligand-binding domain of the thyroid hormone receptor is swapped with the ligand-binding domain of the retinoid acid receptor, the newly formed chimeric receptor will retain the DNA-binding domain of the thyroid hormone receptor and will activate thyroid hormone-responsive genes. However these genes will be activated by retinoid acid via the retinoid acid ligand-binding domain, and not by thyroid hormone.
TF binding can be examined by Electrophoretic mobility shift assay (EMSA). How does this work?
An EMSA is electrophoretic separation of a protein–DNA or protein–RNA mixture on a polyacrylamide or agarose gel for a short period. The speed at which different molecules (and combinations thereof) move through the gel is determined by their size and charge, and to a lesser extent, their shape. The control lane (DNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. However, assuming that the protein is capable of binding to the fragment, the lane with a protein that binds present will contain another band that represents the larger, less mobile complex of nucleic acid probe bound to protein which is ‘shifted’ up on the gel (since it has moved more slowly). SEE NEXT CARD FOR A VISUAL
For those of you who, like me, need a visual easy presentation of EMSA, here you go:
Lane 1 is a negative control, and contains only genetic material. Lane 2 contains protein as well as a DNA fragment that, based on its sequence, does not interact. Lane 3 contains protein and a DNA fragment that does react; the resulting complex is larger, havier, and slower-moving. The pattern shown in lane 3 is the one that would result if all the DNA were bound and no dissociation of complex occurred during electrophoresis. When these conditions are not met a second band might be seen in lane 3 reflecting the presence of free DNA or the dissociation of the DNA-protein complex.
To give you another example, here is an experiment of EMSA with AP-1 binding (remember these had those dimerization domains)
You don’t have to study this
Besides EMSA, TF binding can also be examined with DNAse footprinting. What does this look like?
Can you give a brief overview of how ChIP-seq works?
Most important is that you can show on a gene where TF are
Lastly there was discussed that promoters/enhancers can be examined by a Luciferase reporter essay
I don’t think it’s that important to be honest..
DNA needs to be packaged in (eukaryotic) cells. How is this done?
DNA is tightly associated with histones to form chromatin. A nucleosome is the basic building block of chromatin (147 bp wrapped 1.7x around an octane of histones). Two nucleosomes are linked by H1. The core histones are H2A, H2B, H3 and H4.
For another visual presentation of how DNA is packaged, look at this card.
Since I deem this basic knowledge, I will not discuss it further
